1. Field of the Invention
The present invention relates to the rapid, continuous flow, processing of tissue for histology, from fixation to impregnation. In particular, it relates to an automated tissue processing system that can be operated with continuous throughput and uses a sequential series of different non-aqueous chemical solutions to harden a tissue specimen and to produce a wax-impregnated tissue specimen suitable for embedding and sectioning.
2. Description of the Related Art
Conventional methods prepare tissues for histology by incubation in separate solutions of phosphate-buffered 10% formaldehyde for fixation, a series of increasing concentrations of ethanol for dehydration, and xylene for clearing tissue of dehydration agent, prior to impregnation. Because of the time required for this process, usually 8 hours or longer, it is customary to complete these separate stepsxe2x80x94fixation, dehydration, clearing, and impregnationxe2x80x94overnight in automated mechanical instruments designed for those tasks (see, for example, U.S. Pat. Nos. 3,892,197; 4,141,312; and 5,049,510).
Automated tissue processors implementing such conventional processes are manufactured and sold by, for example, Shandon (HYPERCENTER and PATHCENTRE models), Miles-Sakura (TISSUE-TEK models), and Mopec-Medite (TPC15 model).
A disadvantage of the prior art is that such automated systems have not been capable of continuous throughput. Given the time required to complete tissue processing, cassettes containing tissues are loaded into the system during the day and tissue processing is completed in an overnight cycle. Thus, operation of the prior art systems did not allow tissue-containing cassettes to be processed to completion during the work day.
For example, the TISSUE-TEK vacuum infiltration processor (VIP) series requires more than eight hours for completion of processing. Baskets holding the cassettes are placed in a retort in which tissue is processed. In addition, 14 stations supply solutions of various compositions to the retort. User-programmable software controls this automated process. A rotary valve regulates the movement of solutions between the retort and the various stations; applying pressure or vacuum to the retort when the valve is open causes solution to be pumped out of or pumped into the retort, respectively. Upon completion of a processing run, the instrument automatically prompts the use for a cleaning cycle; this requirement can be overridden only if no paraffin is used. Typically, tissue specimens are batch processed according to the following program:
Typically such conventional methodology demands sending tissue specimens from the operating room, medical office or other sites, to a pathology laboratory sometime during the working day; overnight batch processing of the specimens, so that a tissue specimen suitable for blocking and sectioning is only available on the morning of the next day; and rendering a diagnosis by a pathologist based on microscopic examination of sections prepared from a blocked and sectioned specimen later on that next day (FIG. 1). This requires almost 24 hours between receipt of the specimen and delivery of the pathologist""s report. Although a shortened version of the conventional method is presently practiced, it is feasible only for small biopsies. These biopsies need to be fixed for at least about 30 minutes before initiating the processing cycle. The instrument processing cycle can be programmed to last a minimum of 70 minutes, but is preferably 2 to 2xc2xd hours.
In addition to the minimum one-day delay in giving a surgeon the benefit of a report from the pathologist, there are also problems associated with impeded work flow in the pathology laboratory necessitated by the requisite batch processing of specimens, the safety concerns that attend having instruments operating overnight, the risk of possible instrument failures and the need to monitor the instruments, and the waste of using large volumes of reagents for such processing when automated. Moreover, expensive measures are required to prevent exposure of laboratory personnel to noxious fumes and toxic substances associated with the reagents used in this process. Also, the large volumes of solvent waste and paraffin debris produced by the conventional methodology will pollute the environment if not properly disposed.
Conventional fixation and processing also cause irreversible damage (e.g., hydrolysis of a phosphodiester bond and/or deamidation) to the structure of nucleic acids (e.g., DNA, and especially RNA) that limits the application of genetic techniques for diagnosis and research. Consequently, most DNA and certainly RNA analysis require special precautions with handling of material, such as immediate freezing of fresh tissues to prevent degradation, because retrospective genetic analysis is impaired by the conventional methodology.
Histological diagnosis of a frozen section suffers from multiple disadvantages in comparison to sections prepared from paraffin blocks. U.S. Pat. No. 3,961,097 cautions that the slide prepared from a frozen section xe2x80x9cdoes not possess . . . uniformity of quality;xe2x80x9d xe2x80x9cit is technically more difficult for serial sections of the same specimen to be examined;xe2x80x9d xe2x80x9cextreme caution must be exercised in cutting the specimen in order to ensure a sufficiently thin section and to avoid the possibility of damaging details of the specimen;xe2x80x9d and all the slides must be prepared xe2x80x9cwhile the tissue is in the initial frozen statexe2x80x9d because xe2x80x9c[i]f the tissue is thawed and refrozen for sectioning, it is severely damaged.xe2x80x9d
There is an ever present interest in expediting tissue processing and analysis for diagnostic purposes. Furthermore, recent healthcare focus has been directed to lessening the cost of various procedures including tissue processing. The costs of tissue processing are related to the time for processing and analysis of the specimens, the space required for the personnel and equipment in the laboratory, the volume of reagents (both the purchase price of the pure chemicals and the charges for discarding waste), and the number of personnel required. More importantly, patients and their physicians depend on evaluation and diagnosis by the pathologist to guide treatment. Reducing the amount of time needed to complete tissue processing would lessen the anxiety experienced during the period between obtaining the specimen and delivering the pathologist""s report to the surgeon.
Others have recognized the need to shorten the time required for tissue processing, but they have made only modest improvements in the conventional methods. To accelerate tissue processing, U.S. Pat. Nos. 4,656,047, 4,839,194, and 5,244,787 use microwave energy; U.S. Pat. Nos. 3,961,097 and 5,089,288 use ultrasonic energy; and U.S. Pat. No. 5,023,187 uses infrared energy. U.S. Pat. No. 5,104,640 disclosed a non-aqueous composition of a fixative, a stabilizing agent, and a solubilizing agent that adheres a blood smear to a slide. But the aforementioned patents do not teach or suggest that the entire process of preparing diagnostic tissue slides could be accomplished in less than two hours, starting from fixation and ending with impregnation, with continuous processing of specimens.
Microwave ovens similar in design to those used in home cooking have been used to accelerate the time required for tissue processing. U.S. Pat. No. 4,656,047 claims a method of tissue processing in which at least one of the dehydrating, clearing, or impregnating steps utilizes microwave energy. Fixation may be accomplished by immersing the tissue specimen in chemical fixative and then exposing the specimen to microwave energy for a time sufficient to chemically fix the specimen. U.S. Pat. No. 4,839,194 claims a method of fixing a tissue specimen at a temperature not to exceed 40xc2x0 C. in which the non-thermal effects of microwave energy are used. U.S. Pat. Nos. 4,839,194 and 5,244,787 claim a method of staining tissue specimens utilizing microwave energy.
In such conventional methods of tissue processing, the distribution of microwave energy is not uniform because of reflection and interference effects within the chamber in which the microwaves resonate and the waveguide that conducts the microwave energy from the source to the chamber. U.S. Pat. No. 4,835,354 proposes a mechanical solution utilizing a rotating platform to ensure uniform contact with the microwaves, and mixers and isolaters that disperse and absorb microwaves. U.S. Pat. No. 5,289,140 proposes a solution that utilizes a combination of microwaves of different wavelengths and/or intensities, or sources emitting microwaves of different frequencies. U.S. Pat. No. 5,796,080 discloses adjustable moderating means between the waveguide and a plurality of resonance chambers to individually control the chemical reaction in each chamber, such that the propagated mode of the microwaves in the wave-guide is not substantially changed.
We now describe a microwave unit that provides gentle uniform heating during tissue processing in a manner distinct from that disclosed in the aforementioned patents. Such operation causes minimal damage to the processed tissue, and results in a superior specimen for subsequent histologic studies by a pathologist or cell biologist. In contrast to the solutions disclosed in the patent discussed above, the microwave unit of the present invention does not use a resonance chamber which would be sensitive to the contents of the chamber. This is an important consideration when heating a region that is larger in all dimensions than about 10%-20% of the wavelength of the microwave used and the chemical compositions in the chamber change in different steps of the process. In the invention, microwave energy is distributed into the solution and tissue in such a way as to minimize interference effects. By distributing the energy, it is absorbed by the solution and tissue in one pass through the materials.
Some improvements that result from the invention are summarized here, but other improvements are described below. Convective heat losses from the reaction chamber and the evaporation rate of liquid in the reaction chamber are reduced, volatile substances are prevented from contacting electronic components and vented to protect the laboratory personnel in the vicinity of the unit, errors committed during processing by a human operator are eliminated, the power required by the unit to maintain the liquid temperature in the reaction chamber is reduced, and labor and reagent costs are reduced with this system as compared to manual operation. More subjectively, consistency in the quality of tissue specimens processed by the disclosed process is improved. Although one microwave unit may be used advantageously, multiple units may be operationally and physically linked to accelerate chemical reactions performed in batch and/or continuous mode.
It is an object of the invention to provide a microwave unit and a system for tissue processing that reduces the time required for processing and analysis, and reduces the cost thereof. The tissue processing system is capable of automation and, preferably, accepts specimens in a continuous manner. This allows conversion of existing practice to rapid response surgical pathology for the patient undergoing an operation, and may even allow point-of-care diagnosis by the pathologist in the vicinity of the operating room.
In particular, the microwave unit can provide gentle heating of tissue specimens and prevents over cooking. Uniform heating in the reaction chamber ensures specimens at different locations in the chamber are maintained at about the same temperature. Thus, both the temperature throughout the chamber and during steps of the process are kept substantially the same. A preferred configuration for the chamber is built in whispering gallery mode. Disadvantages of conventional microwave ovens (e.g., hot spots that over cook tissue and do not maintain a solution at substantially the same temperature within the chamber) are avoided by the invention.
The system for tissue processing may utilize the microwave unit as at least one module of the system. Such system may be manually operated or automated. Tissue specimens may be loaded into the system and processed either continuously and/or batchwise. Throughput may also be increased by using a plurality of individual systems arranged in parallel. Continuous processing is accessing an individual series of modules with a tissue specimen or batches thereof prior to the completion of processing without otherwise interrupting the system. The system may be adapted for use in the processes described herein and in previously filed applications; or in other histochemical reactions.
A microwave unit of the invention is comprised of (a) a source for the microwave energy, (b) a waveguide that transmits the microwave energy from the source to a reaction chamber, and (c) a reaction chamber that receives the transmitted microwave energy and processes a tissue specimen by at least initiating hardening (e.g., fixation, dehydration, or a combination thereof). The reaction chamber may contain a plurality of different tissue specimens; for example, the reaction chamber may be configured to contain a carrier or basket loaded with tissue specimens. Preferably, the interior geometry of the reaction chamber is configured to achieve uniform distribution of microwave energy and heating of its contents. Similarly, the source and the waveguide may be configured to achieve minimal energy loss during transmission of the microwave radiation. Power delivered by the microwave source, and thus the heating of the reaction chamber""s contents, may be regulated by a variable current source to allow continuous variation of the power.
The microwave unit may be further comprised of any combination with or without a removable container adapted to fit within the reaction chamber; at least one temperature and/or pressure probe to monitor conditions in the reaction chamber; a closure adapted to fit the reaction chamber and to isolate the reaction chamber from the operator""s surroundings (e.g., a lid attached or removable from the reaction chamber); thermal insulation to retain heat in the reaction chamber; a seal to isolate electronic components from chemicals in the reaction chamber; and control circuitry to receive input from at least one probe and/or timer, and to regulate the microwave energy emanating from the source.
In contrast to the invention, batch processing is required by the prior art because that conventional methodology may take eight hours or longer. In the prior art, specimens are loaded into an automated instrument and cannot be loaded with additional specimens until the entire instrument cycle is completed. All the tissue specimens loaded into the prior art instrument are at the same stage of processing during the entire instrument cycle.
Further advantages of and improvements due to the invention are described below.